1. Introduction
2. Background of the Invention
2.1. Biology of Neurotrophic Factors
2.2. Ciliary Neurotrophic Factor
2.3. Functional Properties of Ciliary Neurotrophic Factor
3. Summary of the Invention
3.1. Abbreviations
4. Description of the Figures
5. Detailed Description of the Invention
5.1. Purification of CNTF
5.2. CNTF Bioassays
5.3. Sequencing of CNTF Protein
5.4. Cloning of CNTF-Encoding DNA
5.5. Expression of a CNTF Gene
5.5.1. Identification and Purification of the Expressed Gene Product
5.6. CNTF Genes and Proteins
5.7. Generation of Anti-CNTF Antibodies
5.8. Utility of the Invention
5.8.1. Diagnostic Applications
5.8.2. Therapeutic Applications
5.8.3. Pharmaceutical compositions
5.8.4. Molecular Probes of the Invention May Be Used to Identify Novel CNTF-Homologous Molecules
6. Example: Molecular Cloning, Expression and Regional Distribution of Rat Ciliary Neurotrophic Factor (CNTF)
6.1. Materials and Methods
6.1.1. Purification and Cleavage of CNTF
6.1.2. Generation of cDNA CNTF Clones
6.1.3. Northern Blot
6.1.4. Expression Of Recombinant CNTF
6.2. Results
6.2.1. Determination of CNTF Amino Acid Sequence
6.2.2. Generation of CNTF cDNA Clones and Sequence Analysis
6.2.3. Expression of Recombinant CNTF
6.2.4. Northern Blot Analysis
6.3. Discussion
7. Example: Expression of CNTF In Escherichia Coli 
7.1. Materials and Methods
7.1.1 Construction of a CNTF Expression Vector
7.1.2 Identification of Bacteria Containing the CNTF Expression Vector
7.2. Results and Discussion
8. Example: Cloning of the Human CNTF Gene
8.1. Materials and Methods
8.1.1. DNA, Plasmid and Phage Vectors
8.1.2. Polymerase Chain Reaction
8.2. Results and Discussion
8.2.1. Evidence for the Existence of a Human CNTF Gene
8.2.2. Cloning of a Fragment of the Human CNTF Gene Amplified by PCR
8.2.3. Cloning of the Human CNTF Gene from a Genomic Library
9. Example: Utility of CNTF-Derived Peptide Fragments
9.1. Materials and Methods
9.1.1. Synthesis of Peptides
9.1.2. Cell Culture
9.1.3. Immunization Protocol
9.2. Results and Discussion
9.2.1. Ability of Antibodies Directed Toward a Synthetic Peptide to Neutralize CNTF Activity
9.2.2. Neurotrophic Activity of a Synthetic CNTF Peptide Fragment
9.2.3. Ability Of Antibodies Directed Toward A Synthetic Peptide To Identify CNTF Containing
10. Example: Ciliary Neurotrophic Factor Promotes Survival of Spinal Cord Neurons
10.1. Materials and Methods
10.1.1. Experimental Animals
10.1.2. Tissue Culture Techniques
10.2. Results and Discussion
10.2.1. Effects of Ciliary Neurotrophic Factor (CNTF) on Mediodorsal (MD) Spinal Cord Neurons
10.2.2. Effects of CNTF on Ventral Spinal Cord Neurons
11. Example: Purified Rat Sciatic Nerve CNTF Prevents Lesion-Induced Cell Death of Motorneurons in the Facial Nerve (VIIth Cranial Nerve) of the Newborn Rat
11.1. Materials And Methods
11.2. Results And Discussion
12. Example: High Level Expression And Purification Of Recombinant Human And Rat Ciliary Neurotrophic Factors In Escherichia Coli 
12.1. Materials And Methods
12.1.1. Bacterial Strains And Plasmids
12.1.1.1. Rat CNTF Vectors
12.1.1.1.1. pRPN11
12.1.1.1.2. pRPN12
12.1.1.3. pRPN37
12.1.1.1.4. pRPN38
12.1.1.2. Human CNTR Vectors
12.1.1.2.1. pRPN32
12.1.1.2.2. pRPN33, PRPN39 pRPN40
12.1.2. Induction Of Protein Synthesis
12.1.3. xe2x80x9cRAPIDxe2x80x9d Protein Extraction
12.1.4. Chromatography
12.1.5. Peptide Analysis
12.1.5.1. Rat CNTF
12.1.5.2. Human CNTF
12.1.6. Biological Activity
12.1.7. Other Methods
12.2. Results And Discussion
12.2.1. Expression Of Rat CNTF
12.2.1.1. Effect Of Copy Number
12.2.1.2. Effect Of Antibiotic Resistance
12.2.2. Expression Of Human CNTF
12.2.3. Purification Of Rat And Human CNTF
12.2.3.1. Yield
12.2.3.2. Characterization
12.2.4. Biological Activity
13. Example: Effects Of Modified And Truncated Ciliary Neurotrophic Factor Protein On Biological Activity
13.1. Materials And Methods
13.1.1. Construction Of Parental Expression Vectors
13.1.2. Construction Of Modified Human Ciliary Neurotrophic Factor Vectors
13.1.3. Construction Of Modified Rat Ciliary Neurotrophic Factor Vectors
13.1.4. Biological Assay Of Ciliary Neurotrophic Factor Activity
13.2. Results And Discussion
14. Example: Additional Effects Of CNTF On Ventral Spinal Cord Neurons
14.1. Materials And Methods
14.1.1. Experimental Animals
14.1.2. Tissue Culture Techniques
14.1.3. Neurofilament (NF) Assay
14.1.4. Choline Acetytransferease (CAT) Assay
14.1.5. Histochemical Staining For Acetylcholinesterase (AchE)
14.1.6. Fractionation Of Ventral Horn Cells by Metrizamide Density Gradient
14.2. Results And Discussion
14.2.1. General Morphologies Of Cultures
14.2.2. Effects Of CNTF On Neurofilament (NF) Levels
14.2.3. Effects Of CNTF On Survival Of AChE-Containing Neurons
14.2.4. Effects Of CNTF In Cat Activity
14.2.5. Delayed Addition Experiment
14.2.6. Effects Of CNTF On Ventral Horn Cultures In The Absence Of Glia
14.2.7. Effects Of CNTF On Metrizamide Gradient-Purified Motorneurons
15. Example: Effect Of Ciliary Neurotrophic Factor On Hippocampal Cultures
15.1. Materials And Methods
15.1.1. Hippocampal Cell Cultures
15.1.2. Assay For GAD Enzyme Activity
15.1.3. Measurement Of Neurofilament Protein
15.1.4. Measurement Of High Affinity GABA Uptake
15.1.5. Immunohistochemical Staining For GAD Or GABA
15.1.6. Immunohistochemical Staining For Neuron-Specific Enolase (NSE)
15.1.7. Histochemical Staining For Calbindin
15.1.8. Histochemical Staining For Acetylcholinesterase
15.1.9. Ciliary Neurotrophic Factor
15.2. Results
15.3. Discussion
16. Example: Novel Monoclonal Antibodies To Ciliary Neurotrophic Factor And A Two-Antibody Sandwich Assay For Human Ciliary Neurotrophic Factor
16.1. Materials And Methods
16.1.1. Generation Of Monoclonal Antibodies To Ciliary Neurotrophic Factor
16.1.1.1. Immunization Protocol
16.1.1.2. Hybridoma Formations
16.1.1.3. Screening Of Hybridomas For CNTF Reactivity
16.1.2. Preparation Of Variants Of Human CNTF
16.1.3. Methodology For Two-Site Immunoassay
16.2. Result And Discussion
17. Ciliary Neurotrophic Factor Promotes Survival Of Spinal Motorneurons In Culture
17.1 Material And Methods
17.1.1. Tissue Culture Techniques
17.1.2. Retrograde Labeling Of Motorneurons And Estimation Of The Purity Of The Culture Of Motorneurons
17.2. Results And Discussion
17.2.1. Effect of Ciliary Neurotrophic Factor (CNTF) On Chick Embryonic Spinal Motorneurons In Culture
17.2.2. Survival Effects of Specific Neurotrophic Molecules and Cytokines
17.2.3. Combination of CNTF, Basic FGF and IGF-I
18. Deposit of Microorganism
The present invention relates to recombinant DNA molecules encoding ciliary neurotrophic factor (CNTF), and to peptides and proteins derived therefrom. The CNTF and related molecules produced according to the invention may be used to treat a variety of neurological disorders.
A number of factors have been identified which influence growth and development in the nervous system. It is believed that these factors may play an important role in sustaining the survival of neuronal populations in the mature, as well as the immature nervous system.
During the normal development of many neuronal populations, there is a defined period of cell death in which many members of the original population die (Hamburger and Levi-Montalcini, 1949, J. Exp. Zool. III:457-501; Hamburger, 1958, Amer. J. Anat. 102:365:410; Hamburger, 1975, J. Comp. Neurol. 160:535-546; Cowan and Wenger, 1968, Z. Exp. Zool., 168:105-124; Rogers and Cowan, 1973, J. Comp. Neurol. 147:291-320; Clarke and Cowan, 1976, J. Comp. Neurol. 167:143-164; Clarke et al., 1976, J. Comp. Neurol. 167:125-142; Hollyday and Hamburger, 1976, J. Comp. Neurol. 170:311-320; Varon and Bunge, 1978, Annu. Rev. Neurosci. 1:327-362; Cowan et al., 1984, Science 225:1258-1265). Neuronal survival has been shown to be proportional to the size of the territory innervated; the smaller the target area of a given neuronal population, the fewer the number of neurons which will survive the period of cell death. It has been suggested that the amount of neurotrophic factor present in the target area may be related to neuronal survival.
Nerve growth factor (NGF) is by far the most fully characterized of these neurotrophic molecules and has been shown, both in vitro and in vivo, to be essential for the survival of sympathetic and neural crest-derived sensory neurons during early development of both chick and rat (Levi-Montalcini and Angeletti, 1963, Develop. Biol. 7:653-659; Levi-Montalcini et al., 1968, Physiol. Rev. 48:524-569). Injections of purified NGF into the developing chick embryo have been found to cause massive hyperplasia and hypertrophy of spinal sensory neurons and sympathetic neurons (Levi-Montalcini and Booker, 1960, Proc. Natl. Acad. Sci. U.S.A. 46:373-384; Hamburger et al., 1981, J. Neurosci. 1:60-71). Conversely, removal or sequestration of endogenous NGF by daily injection of anti-NGF antibodies into neonatal rats has been associated with virtual destruction of the sympathetic nervous system (Levi-Montalcini and Booker, 1960, Proc. Natl. Acad. Sci. U.S.A. 46:384-391; Levi-Montalcini and Angeletti, 1966, Pharmacol. Rev. 18:619-628). Exposure to NGF antibodies even earlier in development either by antibody injections in utero or by passive transplacental transfer of maternal antibodies has been shown to result in a substantial loss of neural crest-derived sensory neurons such as spinal and dorsomedial trigeminal sensory neurons (Goedert et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:1580-1584; Gorin and Johnson, 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5382-5386). Until recently, almost all studies of NGF had focused on its role in the peripheral nervous system, but it now appears that NGF also influences the development and maintenance of specific populations of neurons in the central nervous system (Thoenen et al., 1987, Rev. Physiol. Biochem. Pharmacol. 109:145-178; Whittemore and Seiger, 1987, Brain Res. Rev. 12:439-464).
Neurotrophic factors which have notbeen as well characterized as NGF include brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF).
Ciliary neurotrophic factors (CNTFs) are proteins that are specifically required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervate the ciliary muscle and sphincter pupillae and also smooth muscle present in the choroid layer of the eye.
Ciliary ganglion neurons have been found to be among the neuronal populations which exhibit defined periods of cell death. In the chick ciliary ganglion, half of the neurons present at embryonic day 8 (E8) have been observed to die before E14 (Landmesser and Pilar, 1974, J. Physiol. 241:737-749). During this same time period, ciliary ganglion neurons are forming connections with their target tissues, namely, the ciliary body and the choroid coat of the eye. Landmesser and Pilar (1974, J. Physiol. 241:715-736) observed that removal of an eye prior to the period of cell death results in the nearly complete loss of ciliary ganglion neurons in the ipsilateral ganglion. Conversely, Narayanan and Narayanan (1978, J. Embryol. Ex. Morphol. 44:53-70) observed that, by implanting an additional eye primordium and thereby increasing the amount of available arget tissue, ciliary ganglion neuronal cell death may be ecreased. These results are consistent with the existence of a neurotrophic factor which acts upon ciliary ganglion neurons.
In culture, ciliary ganglion (CG) neurons have been found to require a factor or factors for survival. Ciliary neurotrophic factor(s) (CNTF) activity has been identified in chick muscle cell conditioned media (Bennett and Nurcombe, 1979, Brain Res. 173:543-548; Nishi and Berg, 1979, Nature 277:232-234; Varon et al., 1979, Brain Res. 173:29-45), in muscle extracts (McLennan and Hendry, 1978, Neurosci. Lett. 10:269-273; Bonhady et al., 1980, Neurosci. Lett. 18:197-201), in chick embryo extract (Varon et al., 1979, Brain Res. 173:29-45; Tuttle et al., 1980, Brain Res. 183:161-180), and in medium conditioned by heart cells (Helfand et al., 1976, Dev. Biol. 50:541-547; Helfand et al., 1978, Exp. Cell Res. 113:39-45; for discussion, see also Adler et al., 1979, Science 204:1434-1436 and Barbin et al., 1984, J. Neurochem. 43:1468-1478).
Adler et al. (1979, Science 204:1434-1436) used an assay system based on microwell cultures of CG neurons to demonstrate that a very rich source of CNTF was found in the intraocular target tissues the CG neurons innervate. Out of 8000 trophic units (TU) present in a twelve-day embryo, 2500 TU were found present in eye tissue; activity appeared to be localized in a fraction containing the ciliary body and choroid coat, with a specific activity approximately twenty-fold higher than that found in whole embryo extracts.
Subsequently, Barbin et al. (1984, J. Neurochem. 43:1468-1478) reported a procedure for purifying CNTF from chick embryo eye tissue. CNTF activity was also found to be associated with non-CG tissues, including rat sciatic nerve (Williams et al., 1984, Int. J. Develop. Neurosci 218:460-470). Manthorpe et al. (1986, Brain Res. 367:282-286) reported the purification of mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating CNTF activity from chick eye. In addition, Watters and Hendry (1987, J. Neurochem. 49:705-713) described a method for purifying CNTF activity approximately 20,000-fold from bovine cardiac tissue under non-denaturing conditions using heparin-affinity chromatography. CNTF activity has also been identified in damaged brain tissue (Manthorpe et al., 1983, Brain Res. 267:47-56; Nieto-Sampedro et al., 1983, J. Neurosci. 3:2219-2229).
Carnow et al. (1985, J. Neurosci. 5:1965-1971) and Rudge et al., (1987, Develop. Brain Res. 32:103-110) describe methods for identifying CNTF activity from tissue extracts after blotting cell extracts, separated electrophoretically, onto nitrocellulose paper (Western blotting) and then identifying protein bands containing CNTF activity by inoculating the nitrocellulose with CG neurons and identifying areas of cell survival using vital dyes. These methods were used to determine the apparent molecular weights of the active polypeptides in crude extracts. Using this method, Carnow et al. (1985, J. Neurosci. 5:1965-1971) observed that adult rat sciatic nerve and brain-derived CNTF activities appear to exhibit a different size (24 Kd) than chick CNTF (20.4 Kd).
A number of biologic effects have been ascribed to CNTF although the molecular nature of these activities was not well understood. As discussed above, CNTF was originally described as an activity which supported the survival of neurons of the E8 chick ciliary ganglion, which is a component of the parasympathetic nervous system. A description of other biological properties of preparations known to contain CNTF activity follows:
Saadat et al. (1989, J. Cell Biol. 108:1807-1816) observed that their most highly purified preparation of rat sciatic nerve CNTF induced cholinergic differentiation of newborn rat superior cervical ganglionic neurons in culture. Also, Hoffman (1988, J. Neurochem. 51:109-113) found that CNTF activity derived from chick eye increased the level of choline-O-acetyltransferase activity in retinal monolayer cultures.
Hughes et al. (1988, Nature 335:70-73) studied a population of bipotential glial progenitor cells in the perinatal rat optic nerve and brain; this cell population is believed to give rise to, first, oligodendrocytes and then, second, to type 2 astrocytes. Studies have suggested that oligodendrocyte differentiation occurs from an oligodendrocyte-type 2-astrocyte (O-2A) progenitor cell in the absence of any particular growth factor, whereas type 2 astrocyte differentiation appears to require the presence of a specific inducing protein. Hughes et al. observed that the type 2 astrocyte inducing protein is similar or identical to CNTF (see also Anderson, 1989, Trends Neurosci. 12:83-85).
Heymanns and Unsicker (1987, Proc. Natl. Acad. Sci. U.S.A. 84:7758-7762) observed that high-speed supernatants of neuroblastoma cell extracts produced effects similar to those associated with CNTF activity from chick eye or rat sciatic nerve; the presence of a protein similar but not identical to CNTF (by molecular weight) was indicated.
Ebendal (1987, J. Neurosci. Res. 17:19-24) looked for CNTF activity in a variety of rat and chicken tissues. They observed a fairly wide range of ciliary neuron survival promoting activities among rat, but not chicken, tissues; rat liver, spleen T cells, and submandibular gland cells were found to be associated with low CG survival promoting activity, whereas heart, brain, and skeletal muscle tissues were associated with higher survival promoting activity. Among tissues tested the highest Ciliary Survival promoting activity was observed to be associated with rat kidney.
While the above studies have shown that many tissue and cell extracts contain activities which have similar properties to CNTF, (i.e. they support the survival of E8 chick ciliary ganglion neurons in a tissue culture bioassay), it cannot be assumed that a single or identical protein is responsible for these activities. As shown for the family of fibroblast growth factors (FGFs), for example, a number of distinct polypeptides or protein may possess identical biological activity in a single bioassay.
The neuronal specificity of chick eye and rat sciatic nerve CNTF were initially found to overlap with neuronal populations responsive to NGF. However, distinguishing characteristics between CNTF and NGF became most apparent in studies of the roles of CNTF and NGF in developing neuron populations. Skaper and Varon (1986, Brain Res. 389:39-46) examined the survival requirements of chick dorsal root ganglion (DRG) neurons between embryonic day 6.5 (E6.5) and E15. These DRG neurons, initially responsive only on NGF, were observed to subsequently become responsive to CNTF as well, and eventually appeared increasingly unresponsive to either factor. In addition to differing roles in development, CNTF may also be distinguished from NGF by molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, and by CNTF""s ability to support the in vitro survival of NGF-unresponsive CG neurons (Barbin et al., 1984, J. Neurochem. 43:1468-1478).
The present invention relates to nucleic acid sequences encoding ciliary neurotrophic factor (CNTF) and to the proteins, peptides, and derivatives produced therefrom. In various embodiments of the invention, the nucleic acid sequences, proteins, and peptides of the invention may be used in the treatment of a variety of neurological diseases and disorders, including but not limited to Alzheimer""s disease and Parkinson""s disease.
In additional embodiments, the CNTF nucleic acids, proteins, and peptides of the invention may be used to treat motorneuron diseases, including but not limited to amyotrophic lateral sclerosis (Lou Gehrig""s disease). In a specific embodiment of the invention, CNTF may be used to restore facial nerve function in Bell""s palsy. In a further specific embodiment of the invention, CNTF may be used to support the growth of spinal cord neurons, thereby providing a method of treating spinal cord damage caused by trauma, infarction, infection, nutritional deficiency or toxic agents.
Further, the present invention provides a novel method for producing chemically pure CNTF.
The invention also relates to pharmaceutical compositions comprising effective amounts of CNTF gene products which may be used in the diagnosis and treatment of a variety of neurologial diseases and disorders.
The present invention relates to the cloning, sequencing, and expression of CNTF and provides, for the first time, a means for producing human CNTF utilizing human CNTF-encoding nucleic acid sequences. Furthermore, the CNTF nucleic acid sequences of the invention may be used to identify nucleic acid sequences encoding CNTF or CNTF-homologous molecules in a variety of species and tissues. In additional specific embodiments of the invention, a peptide fragment having CNTF activity has been identified, and antibody to a CNTF peptide that neutralizes CNTF activity has been produced.